Beta Breeding and Genetics at East Lansing, Michigan: Molecular Methods, Genetic Diversity, and Trait Elucidation

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1 Octber-December 2000 Beta Breeding and Genetics at East Lansing 97 Beta Breeding and Genetics at East Lansing, Michigan: Mlecular Methds, Genetic Diversity, and Trait Elucidatin J. Mitchell McGrath, Benild de ls Reyes and Jseph W. Saunders USDA-ARS, 494 Plant and Sil Science Building, Michigan State University, East Lansing, MI ABSTRACT Variety and germplasm develpment and release have been the main gals f the USDA-ARS sugarbeet (Beta vulgaris L.) breeding prgram at East Lansing, MI fr ver 70 years. Prgress has been made in imprving tlerance t Aphanmyces seedling disease, Cercspra leaf spt tlerance, RhizctOllia crwn and rt rt tlerance, herbicide resistance, and lw-tare (smth rt) rt mrphlgy. The genetic basis f these traits is nt well understd, and the current prgram seeks t identify genes that influence the expressin f disease, quality, and mrphlgical traits, and lcate them n beet chrmsmes. The ut-crssing nature fsugarbeet is nt well suited fr largescale genetic analyses. A strategy has been adpted that shuld allw genetic dissectin fa variety f traits thrugh standard genetic analyses. Briefly, a genetic male sterile seed parent that als carries a dminant self-fertility gene is paired with germplasm f interest. The resulting selffertile hybrid is self-pllinated t prduce a segregating F2 ppulatin, which is simultaneusly bserved fr segregatin f targeted traits and tested fr linkage f these traits using mlecular markers. Segregatin f malesterility and self-fertility genes in the F2 gives a range f ptins fr further characterizatin. In additin, this strategy allws relatively rapid intrductin ftraits frm wild and unadapted germplasm while simultaneusly

2 98 Jurnal f Sugar Beet Research Vl 37 N.4 determining the genetic basis fr nvel traits intrduced thrugh such crsses. Using this system will allw systematic explratin f linkage relatin-ships between agrnmic genes frm diverse germplasm surces and mlecular markers. Additinal Key Wrds: breeding systems, germplasm, mlecular markers. Histrical Perspective at East Lansing The USDA has been invlved with sugarbeet at East Lansing, Michigan fr ver 70 years. By J.G. LiJl f the USDA Agricultural Testing Labratry was prviding sugar and purity analyses fr the Farmers and Manufacturers Beet Sugar Assciatin, f Saginaw, MI (Buschlen, 1938). Abut H.L. Khls was hired by Michigan State Cllege t breed and evaluate hybrids fr Michigan (Khls, 1950). Althugh Khls' tenure in sugarbeet was relatively shrt. it is ntable that he created Michigan Hybrid 18. VF. Savitsky reprted recvering tw plants with mngerm seed frm a 1.6 hectare seed prductin field f Michigan Hybrid 18 in Oregn (McFarlane, 1993; Savitsky, 1950; Oldemeyer, 1998). Shrtly after WWII, GJ. Hgabam assumed agrnmic testing and breeding respnsibilities fr the USDA at East Lansing, until he retired in Hgabam cllabrated in develping germplasm with tlerance t Aphanmyces seed ling disease, Rhiwctnia crwn and rt rt, Cercspra leaf spt, as well as mngerm and CMS / O-type, resulting in the wide use f US H20 (Ce and Hgabam, 1971) and ther varieties in Michigan. Beginning in J.W. Saunders intrduced a bitechnlgical perspective t the prgram, initially fcusing n sht culture fr clnal prpagatin and sht regeneratin frm callus (Saunders, 1998), which has expanded t include smatic embrygenesis (Saunders and Tsai. 1999), and fr a time, smatic cell se lectin fr herbicide resistance (Saunders et a1.,1992). Frm 1982 t J.e. Theurer develped smth-rt, lw tare gentypes in cmbinatin with imprved agrnmic perfrmance and increased disease tlerance (Saunders et a/., 1999). J.M. McGrath assumed duties as a USDA Research Geneticist f r sugarbeet breeding and genetics in 1996, in part t cntinue prir activities but als t expand the fcus f the prgram by applying mlecular genetics t sugarbeet imprvement. Current Fcus and Prgram Develpment The missin f the USDA Agricultural Research Service is t prvide practical slutins t agricultural prblems f natinal scpe. Such slutins ften require lng-term cmmitments and high-risk appraches.

3 Octber-December 2000 Bela Breeding and Genetics at East Lansing 99 Sugarbeet imprvement is an area where the USDA-ARS has had a pivtal rle. The East Lansing prgram has three main bjectives: a) t cntinue the germplasm enhancement activities initiated in earlier years, b) t examine the genetics f agrnmic and disease resistance traits, and c) t examine prblems femergence and stand establishment. Each f these is inter-related, and the fcus in this paper is strategies bei!1g used t satisfy the secnd bjective. Beet has been eaten by humans fr thusands f years, either as a leafy vegetable r fleshy rt (Frd-Llyd and Williams, 1975). Wild beet, and related species, are endemic thrughut the Mediterranean regin and the maritime regins f Western and Nrthern Eurpe. Fr sugar, beet has a histry f abut 200 years, and much f the increase in sucrse percentage frm initial t current levels ccurred within the first 100 years (Fischer, 1989; Cke and Sctt, 1993). Ersin f genetic diversity in sugarbeet has been a cncern, since: a) sugarbeet has been selected frm a narrw funding ppulatin, b) selectin pressure fr increased sucrse cntent has been intense, and c) a limited number f varieties frmed the riginal germplasm base f breeding prgrams in the U.S. and elsewhere, riginally intrduced frm Eurpe. Als, recessive genes fr expressin f mngerm seed and cytplasmic male sterility are nearly universal in mdern cultivars, and this has impsed an additinal genetic bttleneck (Bsemark, 1993), as well as resulted in cytplasmic unifrmity in cmmercial hybrids wrldwide. Genetic bttlenecks during sugarbeet breeding have nt necessarily impacted genetic diversity as whle, since breeding prgrams have generally maintained allelic di versity by virtue f ppulatin imprvement appraches used during mst f the crp's histry. Selectin ver the past 80 years has sub-divided the genetic diversity int individual breeding ppulatins, at least amng germplasm releases in the U.S. (McGrath et a!., 1999). Genetic diversity in sugarbeet is clearly much less than in its wild prgenitrs, and it is likely that useful characters (e.g. disease resistances, seedling vigr, nutrient efficiency, phtsynthate partitining, etc.) may be extracted frm accessins available thrugh natinal and internatinal germplasm cnservatin rganizatins. Determining the genetic basis f a wide range f agrnmic traits and expanding the number and types f agrnmic genes available t sugarbeet breeders is a daunting task. Discvering genes fr agrnmic traits culd be cmbined with deplyment f these genes in enhanced germplasm, and significant benefits may result in a mdest timeframe. Shifting away frm sme f the traditinal breeding methds is required t merge gene discvery and deplyment. New appraches shuld be flexible enugh t be systematically applied t a large number f trait and

4 loa Jurnal f Sugnr Beet Research Vl 37 N.4 germplasm surces. A majr limitatin fr genetic analyses in beet is the presence f a cmplex self-incmpatibility system (Lundqvist et ai., 1973), and this generally limits generating sufficient seed f a single hybrid individual necessary t determine segregatin patterns. T circumvent this limitatin, the dminant self-fertility gene (Owen, 1942) is used as a central part f the East Lansing prgram. Using enfrced selfing, a large number f segregating ppulatins can be created frm crsses f single individuals, ne f which has the self-fertility gene, by placing pllen-prf bags n cnfirmed hybrids at flwering. Enfrcing crssing between parents relies n a recessive nuclear male-sterility gene that ensures all seed prduced n the mther plant are hybrid. Thrugh tl1is apprach, ver 50 hybrid cmbinatins have been made and ver 500 segregating ppulatins generated in the past tw years. An advantage f the self-fertility / male-sterility (Sf/ ms) apprach is the range f genetic materials generated. Mlecular marker analyses fit well with this scheme as each F, ppulatin is derived frm a single F[ hybrid plant. With a marker set cvering the genme at a reasnable density, each ppulatin can be gentyped. The marker infrmatin is used t develp hyptheses n the inheritance f traits via QTL-type appraches (Tanksley and Nelsn, 1996), and these hyptheses can be verified in F} r back-crss ppulatins derived frm selected F2 individuals. A disadvantage f the Sf / ms apprach is that segregatin distrtin will ccur at and near the self-incmpatibility lcus (lci) ifne f the parents is self-incmpatible. This is because the gametphytic incmpatibility system f beet will arrest grwth f pllen tubes expressing the same self-incmpatibility allele, such as wuld be expected during self-pllinatin. Culture f excised vules frm hybrids may allw recvery f self-sterile plants if they are desired fr ppulatin imprvement breeding appraches. Ideally, mlecular markers fr a trait shuld be genes invlved in the expressin f that trait. Current knwledge is incmplete regarding the numbers, lcatins, and gene actin fr mst sugarbeet characters. Until this infrmatin is available, expressed genes in general may be a gd substitute. We have begun t sequence expressed genes, with the aim t develp Expressed Sequence Tags (ESTs) fr germplasm evaluatin and characterizatin. Sequenced cdna clnes are currently used as RFLP markers, with PCR-based strategies t be develped frm cdna nucletide sequences and the genetic lci they detect. T date, we have sequenced ver 500 randmly islated cdna clnes (Table 1) frm tw libraries. These are searched fr sequence similarity t previusly characterized genes, and inspected fr 'candidate' genes presumed t be invlved in the expressin f a trait. Sucrse metablism genes frm many rganisms have been have been clned, fr example, and Schneider et al. (1999)

5 Table 1. Sugarbeet gene prducts identified thrugh nucletide sequencing and database searching. Number Functinal Class f ESTs Examples (e< 10 1 ) Amin Acid Metablism 36 (6.5%) Glutamine synthetase; Aspartate amintransferase; Phsphserine amintransferase; Alanine amintransferase; S-Adensylmethinine synthetase; S-Adensyl hmcysteinease; Ornithine amintransferase Carbhydrate Metablism 15 (2.7%) alpha-amylase; beta-amylase, 1-0-dexyxyllulse 5-phsphate synthase, UO P -gl ucurnl y Itransferase, cellulase, GO P- man nse pyrphsphry lase Mnsaccharide Metablism 28 (5.1 %) Fructse I,6-bisphsphatase; Malate dehydrgenase; Phsphfructkinase; Glyceraldehyde 3-phsphate dehydrgenase; Phsphglucnate dehydrgenase; Aldlase; Enlase; Sucrse-phsphate synthase Lipid Metablism 9 (1.6%) N-Acetylglucsaminyl-phsphatidylinsitl bisynthetic prtein; N myristyltransferase; AcetylCA carbxylase; Fatty acid hydrxylase Electrn Transprt 5 (0.9%) Cytchrme c; Ubiquinl-cytchrme c reductase; Ubiquinne Oxidreductase Phtsynthesis 8 (1.5%) RUBISCO; Mndehydrascrbate reductase; Phtsystem II 10 kd prtein (cntinued n next page) () <) 0 () 3 0.., N I:!:! '" is t:rl 0.. '" fio '" '"0.. Cl. V> rn OJ r-' OJ en 5 (JO

6 Table 1 (cntinued). Sugarbeet gene prducts identified thrugh nucletide sequencing and database searching. Number Functinal Class f ESTs Examples (e < 10 1 ) Membrane Transprt 29 (5.3%) Ca:?+ transprting ATPase; H+ transprting ATPase; Cu 2 + transprting ATPase; Ptassium transprt prtein; ABC-type transprt prtein; Prin; Amin acid/peptide transprter; Na+ /H+ exchanger prtein; Vacular H+ ATPase; Oxglutarate/Malate trans lcatr prtein Secndary Metablism 14 (2.5%) Sesquiterpene cyclase; Raucaffricine-O-beta-D-glucsidase; Pectinesterase; Caffeyl-CA O-methyltransferase Replicatin 3 (0. 1%) DNA plymerase; DNA gyrase Transcriptin Factr IS (2.7%) AP2 dmain-cntaining prtein; DREB2A; Myb-related prteins; bzip transcriptin factr tv '-< c.., ::l 0> ọ.,., VJ cr c e; c (j) (j) en '" (j) 0> ri :0 Translatin 26 (4.7%) Elngatin factrs (EFI-alpha; Tu); Initiatin factrs (EIF2, EIF4A, EIF5A) Histnes 8 (1.5%) H2B; H3; H4 Signal Transductin/ Prtein Kinase (cntinued n next page) 23 (4.2%) Calmdulin; Guanylate nucletide binding prtein; Serine-Threnine prtein kinase; Receptr prtein kinase; Mitgen-activated prtein kinase 2: w -.l Z? +>

7 Table} (cntinued). Sugarbeet gene prducts identified thrugh nucletide sequencing and database searching. Number Functinal Class f ESTs Examples (e < 1OJ) Envirnmental Stress-Related 43 (7.8%) Drught & salt inducible prteins (RAN 1, HAL3A, Di 19); Heat shck prteins (HSP70, HSP80. HSP90); Betaine-aldehyde dehydrgenase; Alchl dehydrgenase; PR-prtein (chitinase; PR3); Trehalse-6-phsphate synthase; Aluminum-induced prtein Ribsmal Prtein 68 (12.3%) 40S, 50S, 60S Miscellaneus 77 (14%) Actin: Tubulin; Ubiquitin; Extensin; Elastin: Nuclelin; Ubiquitin cnjugating enzyme; ATP Synthase; Prteasme assembly prteins; Phytase; AminacyltRNA synthetase; Histne deacetylase; RNAse: Maturase; Nucleside triphsphatase; Inrganic pyrphsphatase; Glutathine S-transferase Unknwn 175(31.8%) Ttal 55} () S (b b (b () (b a 0 0:> :s '" (b Q. C/O 0.> Q. (b n' '" t11 0> t"'"' 0> '" S' C/O 8

8 104 Jurnal f Sugar Beet Research Vl 37 NO.4 are using these as candidate genes invlved in sucrse accumulatin. Other strategies such as differential mrna display and representatinal difference analysis (Liang and Pardee, 1992; Lisitsyn et a!., 1995) may be useful in identifying differentially expressed genes, such as stress-induced genes during germinatin (de ls Reyes and McGrath, unpublished). Given an infrmative set f genetic markers, current technlgy wuld allw rapidly gentyping individuals in ppulatins. Mlecular marker prfiles fr each plant in a breeding ppulatin culd be dne prir t agrnmic evaluatin, if the current technlgies were develped fr beet. It is unlikely that any single ppulatin is ging t detect all genes invlved in trait expressin. Hwever, the cmbined pwer f multiple ppulatins, sites, and years will identify mst if nt all genes r alleles that cntribute t quantitative traits. Integrating this infrmatin will require a cmmn set f markers that are unifrmly distributed thrughut the genic poltin f the genme, and nes that are easily assayed with minimal cst. Analyzing the cmbined data sets will be challenging, and cmputatinal algrithms using artificial intelligence, fuzzy lgic, and data mining appraches, fr example, will need t be develped. Marker tags alne are f little value t the grwer r prcessr, s strategies t rapidly screen agrnmic perfrmance f segregating ppulatins will need t be implemented. Scring ppulatins fr agrnmic traits is a significant undertaking that is ften labr-intensive and subject t envirnmental variability. Imaging analysis and remte sensing are just beginning t be applied t agriculture and may shw prmise in scring traits. Imaging can analyze each plant individually, and make repeated measures f single plants ver time (e.g. nn-destructi ve testing), if munted n a mbile platfrm. An applicatin wuld fllw the curse f field emergence ver time, and perhaps pinpint critical aspects amenable fr a targeted breeding effrt. Imaging can als shw relative grwth rates, leaf area indices, and canpy cver changes during the grwing seasn. Cercspra leaf spt disease prgressin may be assessed with the same equipment. Other applicatins may be spectral analyses (hyper-spectral imaging) as early indicatrs f plant stress. Near-infrared reflectance spectrscpy may allw identificatin f specific mlecules such as secndary metablites and sucrse. Such capability wuld allw fr high-thrughput agrnmic evaluatin (e.g. screening large ppulatins cnsistently and rapidly), at least as a first apprximatin, which wuld help t refine breeding and selectin decisins. Such activities are beynd the current scpe and expertise f the USDA at East Lansing, but cllabratins t achieve these gals shuld be fstered. In summary, the emphasis f USDA prgram at East Lansing is shifting twards integratin f bitechnlgies fr sugarbeet imprvement.

9 Octber-December 2000 Beta Breeding and Genetics at East Lansing 105 A strategy has been develped which will be tested ver the cming years. Many pprtunities fr cllabratin exist nw and in the future (as they have in the past), and we appreciate the supprt f the sugar cmmunity in fstering past and future achievements. LITERATURE CITED Bsemark, N.O Genetics and breeding. p In D.A. Cke and RK. Sctt. (ed.) The sligar beet crp: Science int practice. Chapman and Hall, Lndn. Buschlen, M.J Beet variety trials in Michigan, Ohi, Indiana area. Prc. Am. Sc. Sugar Beet Techn!. I: Ce, G.E. and G.J. Hgabam. J971. Registratin f US H20 sugarbeet. Crp Sci. 11: 942. Cke, D.A. and RK. Sctt Intrductin. p. xiv-xix. In D.A. Cke and RK. Sctt, (ed.) The sugar beet crp: Science int practice. Chapman and Hall, Lndn. Fischer, H.E Origin f the 'Weisse Schlesische RUbe' (white Silesian beet) and resynthesis f sugar beet. Euphytica 41: Frd-Llyd, B.Y. and J.T. Williams A revisin f Beta sectin Vulgares (Chenpdiaceae), with new light n the rigin f cultivated beets. Bt. J. Linn. Sc. 71: Khls, H.L A genetic study f 17 FI hybrids and their inbred parents. Prc. Am. Sc. Sugar Beet Techn!. 6: Liang, P. and A. Pardee Differential display f eukarytic messenger RNA by means f plymerase chain reactin. Science 257: Lisitsyn, N., N. Lisitsyn, and M. Wigler Clning the differences between tw cmplex genmes. Science 259: Lundqvist, A., U. 0sterbye, K. Larsen, and, I. Linde-Laursen Cmplex self-incmpatibility systems in Ranunculus acris L. and Beta vulgaris L. Hereditas 74:

10 106 lurnal f Sugar Beet Research Vl 37 N.4 McFarlane,l.S The Savitsky stry. 1. Sugar Beet Res. 30: McGrath, 1.M., e.a. Derric, and Y. Yu Genetic diversity in selected, histrical USDA sugarbeet germplasm releases and Beta vulgaris ssp. maritima. Ther. App!. Genet. 98: Oldemeyer, R White gld mining: mngerm sugarbeet seed, a saga, finis. Sugar J. May, p.7. Owen, F.V Inheritance f crss- and self-sterility and self-fertility in Beta vulgaris. J. Agric. Res. 64: Saunders, 1.W Registratin f REL-l and REL-2 sugarbeet germplasms fr tissue culture genetic manipulatins. Crp Sci. 38: Saunders, 1.W., G. Acquaah, K.A. Renner, and W.P. Oley Mngenic dminant sulfnylurea resistance in sugarbeet frm smatic cell selectin. Crp Sci. 32: Saunders, 1.W., 1.M. McGrath, 1.M. Hallin, and 1.e. Theurer Registratin f SR94 sugarbeet germplasm with smth rt. Crp Sci. 39: 297. Saunders, 1.W. and C.J. Tsai Prductin f smatic embrys and shts frm sugarbeet callus: Effects f abscisic acid, ther grwth regulatrs, nitrgen surce, sucrse cncentratin and gentype. In Vitr Cel!. Deve!. Bi!. -Plants 35: Savitsky, VF Mngerm sugar beets in the United States. Prc. Am. Sc. Sugar Beet Techn!. 6: Schneider, K., D.C. Brchardt, R. Schafer-Pregl, N. Nagl, e. Glass, A. leppssn, C. Gebhardt, and F. Salamini PCR-based clning and segregattin analysis f functinal gene hmlgues in Beta vulgaris. Ml. Gen. Genet. 262: Tanksley, S.D. and J.e. Nelsn Advanced backcrss QTL analysis: a methd fr the simultaneus discvery and transfer f valuable QTLs frm unadapted germplasm int elite breeding lines. Ther. App!. Genet. 92: